Lung endothelium forms a semi-permeable barrier that restricts water, solute and macromolecular access to tissue compartments. Inflammatory agents promote calcium influx across the endothelial cell plasma membrane, which triggers cytoskeletal reorganization, induces inter-cellular gaps, and increases permeability. However, endothelial cells express many different ion channels, bringing into question exactly which channel(s) provides the calcium source responsible for this cytoskeletal response. We have recently identified an endogenously expressed channel that is activated by inflammatory mediators, and provides a calcium source that induces inter-endothelial cell gaps. This channel is comprised of three different transient receptor potential proteins from the canonical sub-family (TRPC), including one TRPC1, one TRPC3 and two TRPC4 subunits. Patch clamp studies reveal that activation of the TRPC1/3/4 channel results in a calcium selective current, referred to as ISOC. The mechanism necessary to confer calcium selectivity to the TRPC1/3/4 channel is unknown. Our preliminary data indicate that Orai1 functions as an ancillary subunit, and is necessary to confer calcium selectivity to this channel. TRPC1/3/4 calcium selectivity may represent a physiologically important feature of the channel, as it impacts how much calcium permeates the channel and reaches the cortical actin rim. The TRPC4 subunits within this channel bind to protein 4.1, and protein 4.1 tethers the channel to the spectrin membrane skeleton. Spectrin directly binds to actin, and this interaction is necessary for proper alignment of the cortical actin rim. Indeed, increased calcium disrupts the spectrin-actin interaction, suggesting calcium that permeates the TRPC1/3/4 channel may disrupt spectrin-actin binding and catalyze gap formation. Thus, this proposal tests the overall HYPOTHESIS that Orai1 interacts with TRPC1/3/4 and establishes the channel's calcium selectivity, which is necessary to increase membrane calcium to concentrations that disrupt the spectrin-actin interaction causing inter-endothelial cell gap formation. Specific aims test the related hypotheses that: [1] Orai1 constitutively interacts with TRPC1/3/4, and contributes to channel calcium selectivity; [2] activation of the TRPC1/3/4 channel requires an intact protein 4.1-TRPC4-Orai1 interaction; [3] calcium permeation through the TRPC1/3/4 channel disrupts the f-actin- spectrin interaction resulting in cytoskeleton reorganization and increased permeability. Studies proposed in this application address three fundamental, yet poorly understood areas in calcium signaling and endothelial cell biology. First, we will address whether Orai1 interacts with the TRPC1/3/4 channel. Second, we will address the mechanism responsible for determining TRPC1/3/4 calcium selectivity. Third, we will determine the physiological importance of TRPC1/3/4 calcium selectivity.